Alzheimer's Disease (AD) is a major source of suffering, disability and societal costs that currently lacks effective treatment options. Variants in lipid metabolism genes are among the strongest risk factors for late-onset AD. However, the specific mediators and mechanisms linking dysfunctional lipid metabolism to AD pathogenesis in humans remain to be identified. Our Unit seeks to fill this gap by systematically characterizing lipid-related abnormalities throughout the full spectrum of AD, with the long-term goal of identifying new therapeutic leads and new approaches for preventing or treating AD and related neurodegenerative diseases. Approach With these goals in mind, we are developing and applying advanced multiplex immunohistochemistry protocols to simultaneously label >100 cellular and molecular markers in rapidly collected and rapidly processed human medial temporal lobe specimens representing the full spectrum of AD-type pathology (from healthy aging to Mild Cognitive Impairment (MCI) to AD), in collaboration with D. Maric (Intramural NINDS). The spatial resolution and breadth of markers afforded by this approach are advancements over traditional approaches which typically provide either a much more limited set of markers, or lack the spatial resolution and context provided by concurrent labeling of cellular and architectural markers. Cellular and molecular markers included in this project can be classified into four general categories: (1) cellular and molecular architectural (non-pathological) markers; (2) classic AD-type pathological markers (i.e. amyloid oligomers and plaques, truncated and hyperphosphorylated tau); (3) emerging AD-type pathological markers (i.e. immune abnormalities, mitochondrial dysfunction, senescence); and (4) a broad range of markers to delineate lipid-related molecular pathways and derangements. Accomplishments Over the past seven months we have made major progress in designing and implementing a rigorous antibody validation protocol and several multi-round staining protocols, and have successfully completed two pilot multiplex immunostaining phases. In Phase 1 we successfully stained 40 cellular and molecular markers in AD specimens. In Phase 2, we successfully stained >70 markers in three cases (Control vs. MCI vs. AD), including an expanded set of validated lipid metabolism and neurodegeneration markers. We are currently working toward the goal of >100 validated markers representing the 4 categories above. In the next Phase we will stain the full set of 30 cases and apply both traditional image analysis techniques and sophisticated artificial intelligence approaches to analyze the images and data. Summary This project will provide high-quality, quantitative data and high-resolution images depicting lipid-related metabolic pathways and derangements throughout the full spectrum of AD progression, in the context of CNS cytoarchitecture and more established AD-type pathologies. In addition, the construction and validation of CNS antibody panels and protocols used for this project will provide a plug-and-play template allowing for comprehensive assessment of CNS pathological markers that can be applied to other tissues and diseases in future projects.